Integrated fluorescence analysis system

ABSTRACT

An integrated fluorescence analysis system enables a component part of a sample to be virtually sorted within a sample volume after a spectrum of the component part has been identified from a fluorescence spectrum of the entire sample in a flow cytometer. Birefringent optics enables the entire spectrum to be resolved into a set of numbers representing the intensity of spectral components of the spectrum. One or more spectral components are selected to program a scanning laser microscope, preferably a confocal microscope, whereby the spectrum from individual pixels or voxels in the sample can be compared. Individual pixels or voxels containing the selected spectral components are identified and an image may be formed to show the morphology of the sample with respect to only those components having the selected spectral components. There is no need for any physical sorting of the sample components to obtain the morphological information.

BACKGROUND OF INVENTION

This invention relates to fluorescence imaging and, more particularly,to selective fluorescence imaging using fluorescence spectral propertiespreselected by flow cytometer analysis.

Fluorochromes are frequently bound to molecular cell components to yieldquantitative information about the cell components. Several differentfluorochromes may be present in a given cell sample to yield a number ofemission spectra. Further, the emission spectrum from a givenfluorochrome may be altered as a result of changes in the physiologicalstate of the cells.

Fluorescence is generally detected by one or more detectors that areresponsive to specific wavelengths of interest. Cytometry, either flowcytometry or image cytometry, may use this fluorescence as some measureof individual cells or cellular constituents. Cytometry candifferentiate between effects that affect all cells equally and effectsthat cause subpopulations of cells within the population to change. Seegenerally Clinical Cytometry, M. Andreeff ed., New York Academy ofSciences, New York, N. Y. (1986), incorporated herein by reference. Itwill be appreciated that an increasing number of fluorochromes withdifferent sensitivities to changes in cell physiology and labeledmonoclonal antibodies that recognize specific antigens impose arequirement for an increasing number of spectral channels forfluorescence analysis.

U.S. Pat. No. 4,905,169, issued Feb. 27, 1990, to Buican et al.,incorporated herein by reference, teaches cytometry apparatus, andparticularly flow cytometry apparatus, for simultaneously resolving aplurality of spectral properties from a total fluorescence spectrum. AFourier-Transform (FT) spectrometer encodes spectral information in awaveform (interferogram) on which it then performs a numerical, ratherthan optical, spectral analysis. When installed in a flow cytometer, theFT spectrometer analyzes the total fluorescence emitted by singleparticles as they cross an excitation laser beam and resolves thisemission into a set of separate fluorescences, each described by acharacteristic emission spectrum which has been selected by theoperator. Thus, a set of numbers (spectral parameters) is produced foreach particle, describing the spectral properties of the individualparticles in the sample. Taken separately, each spectral parameterrepresents an enhancement of a specific fluorescence, since it isobtained by eliminating all other emission spectra from the totalfluorescence.

Image cytometry devices are also used for detecting fluorochromes inbiological specimens and may be implemented as a laser-scanning confocalmicroscope. See generally Handbook of Biological Confocal Microscopy, J.Pawley ed., Plenum Press, New York, N.Y. (1989, 1990), incorporatedherein by reference. However, fluorescence data is obtained on apixel-by-pixel basis over the biological sample and it is difficult toquantify specific cell subpopulations in the sample. This is normallydone by physically sorting a selected subpopulation by flow sorting,recovering the cells, and placing them on a slide for morphologicalanalyses. Accordingly, the present invention incorporatesFourier-Transform technology into the laser-scanning confocal microscopeto enable image enhancement for a selected superposition of fluorochromespectra to effect a virtual sorting of a subpopulation previouslyidentified.

It is an object of the present invention to generate concurrent multipleimages from a laser-scanning confocal microscope, each enhanced for aparticular emission spectrum.

It is another object of the present invention to generate an image froma biological specimen enhanced for an emission spectrum selected fromflow cytometer data.

One other object of the present invention is to enable a virtual sortingof cells in a biological sample without physical sorting.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

SUMMARY OF THE INVENTION

To achieve the foregoing and other objects, and in accordance with thepurposes of the present invention, as embodied and broadly describedherein, the apparatus of this invention may comprise an integratedfluorescence analysis system. A flow cytometer is provided withbirefringent optics for simultaneously measuring a first plurality ofspectral wavelengths present in a first fluorescence spectrum from afirst sample. The fluorescence spectrum is resolved by a first processormeans into first numbers representing the intensity of spectralcomponents of the first plurality of spectral wavelengths. An imagingcytometer is also provided with birefringent optics for simultaneouslymeasuring a second plurality of spectral wavelengths present in a secondfluorescence spectrum from a second sample. A second processor meansthen resolves the second fluorescence spectrum into second numbersrepresenting the intensity of spectral components of the secondplurality of spectral wavelengths. Communication means connects thefirst processor means with the second processor means so that the firstor second numbers can be input to the second or first processor means,respectively, for enhancing spectral components of the second or firstsamples, respectively.

In another characterization of the present invention, a programmablespectral imaging cytometer includes microscope means for scanning alaser beam over a selected sample image plane to excite a fluorescencespectrum over the image plane. Birefringent optics resolves thefluorescence spectrum into a plurality of pixels, each pixel having apixel fluorescence with one or more spectral wavelengths. Processormeans resolves each pixel fluorescence into numbers representing theintensity of spectral components in the pixel fluorescence. Numbers arestored representing spectral components from a preselected spectrum. Aselection means, such as a computer, then selects those pixels havingnumbers corresponding to the stored numbers to develop a pix pixel imageover the image plane corresponding to the preselected spectrum.

In one other characterization of the present invention, a methodprovides for obtaining from an actual sample a sample representationhaving preselected spectral components. The actual sample is irradiatedwith a laser beam to stimulate a fluorescence spectrum from the samplehaving one or more spectral wavelengths. The fluorescence spectrum isresolved into sample numbers representing the intensity of spectralcomponents in the fluorescence spectrum. The sample numbers are comparedwith stored numbers representing the preselected spectral components.Portions of the sample are then identified that have a fluorescencespectrum corresponding with the preselected spectral components.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand, together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1 is a schematic in block diagram form of an integratedfluorescence analysis system according to the present inventionparticularly showing flow cytometry components.

FIG. 2 is a schematic in block diagram form of an integratedfluorescence analysis system according to the present inventionparticularly showing imaging cytometry components.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a sample is irradiated with alaser beam to produce a fluorescence response spectrum having aplurality of wavelengths. Birefringent optics, as described in U.S. Pat.4,905,169, incorporated by reference, enable a Fourier analysis toresolve the fluorescence spectrum into numbers representing theintensity of each of the spectral wavelengths of the fluorescence. Thus,a set of numbers representing base spectra can be selected from thetotal spectrum and thereafter used to provide virtual sorting ofbiological particles against the base spectra.

As shown in FIGS. 1 and 2, an integrated fluorescence analysis systemincorporates a flow cytometer 10 and an imaging cytometer 20, each withbirefringent optics, and communicating with one another through acommunication channel, such as computer 52. Flow cytometer 10 andimaging cytometer 20 cooperate in the following ways: (a) base spectrafor resolving total cell/pixel fluorescence are shared; and (b) thespectral data obtained by one instrument are used to program the otherinstrument. Then, if the analysis of a cell suspension reveals theexistence of a spectrally distinct subpopulation, the average spectrumfor that population can be transferred from one instrument to the otherto enhance the cell/pixel data and identify cells/pixels that have thesame spectral properties. In the imaging cytometer, an image can then beformed with only those pixels containing spectral features of interest.Conversely, the average spectrum of a whole cell of interest, or that ofa particular morphological detail, can be transferred to the flowcytometer, where it is used to identify and quantitate cellsubpopulations with the same spectral properties.

Referring now to FIG. 1, flow cytometer 10 communicates with imagingcytometer 20 through computer 52. Flow cytometer 10 provides forsimultaneously measuring a plurality of spectral wavelengths present influorescence produced when a laser beam 12 excites sample materials,such as biological cells, contained in focused flow stream 11. Theoutput fluorescence is input by lens 14 to birefringent interferometer16, as particularly described in the '169 patent. It will be appreciatedthat the sample materials may be biological particles such aschromosomes or other cellular material. The particles can be unstained,wherein the fluorescence is intrinsic fluorescence or autofluorescence,or stained with one or more fluorochromes.

Interferometer 16 includes birefringent elements and polarizing elementsto introduce a time-varying phase difference between spectral componentsof the output fluorescence. Detector 22, which may be a photomultipliertube (PMT), produces an electrical output signal characteristic of thesum of the intensities of the spectral components incident on detector22. Analog-to-digital (ADC) converter 26 digitizes the electricalsignals output from detector 22 for input to array processor 28. Clock24 is phase-locked with interferometer 16 and synchronizes operation ofthe system elements. Clock 24 strobes ADC 26, processor 28, and storagearray 32 at a frequency that is greater than the frequency at whichbirefringent interferometer 16 is driven.

Array processor 28 receives the output from ADC 26 for processing.Processor 28 includes a multiplicity of processors equal to the numberof spectral channels over which the fluorescence is to be measured. Eachprocessor computes and outputs a single number representing theintensity of one spectral channel, or wavelength, of the fluorescencefrom samples in flow stream 11. Storage array 32 then stores thecomputed spectral numbers. Computer 52 enables imaging cytometer 20 tocommunicate with storage array 32, as hereinafter discussed.

Referring now to FIG. 2, there is shown a block diagram schematic of animaging cytometer according to the present invention. It will beunderstood that the optical analyzing elements, i.e., birefringentinterferometer 16', lens 18', detector 22', array processor 28', andclock 24' are identical with the corresponding components 16, 18, 22,28, and 24 discussed in FIG. 1. A scanning laser microscope 38, 40, 42,44, and 46 provides the optical input to interferometer 16'. A confocalscanning microscope, e.g., a BioRad MRC-500, can scan over a pluralityof image planes within sample 36 to develop a three dimensional image.

Laser beam 38 is input through dichroic mirror 42 to scanning mirror 44,which is driven by scan driver 40 to traverse beam 38 through optics 46over an image plane within specimen 36. Fluorescence excited fromspecimen 36 is transmitted to interferometer 16' through microscope lens46, scanning mirror 44, and dichroic mirror 42. The fluorescence datafrom specimen 36 is provided as pixel information for a given imageplane, i.e., scan driver 40 is synchronized through clock 24' so thatfluorescence data are obtained from a sequence of discrete locationsover the image plane within specimen 36. It will be appreciated that thefluorescence spectrum from each pixel will be a composite spectrum,i.e., one or more wavelengths, from the fluorescent components in thepixel area. It will also be appreciated that the confocal microscope canscan image planes through the entire volume of a sample so that each"pixel" represents a volume, or "voxel.+ The term "pixel" will be usedherein to mean a focal region for scanning microscope 45, whether on asurface area or within a sample volume.

The output from array processor 28' is a set of numbers representing thespectral components of each pixel fluorescence. In one embodiment of thepresent invention, computer 52 communicates with flow cytometer 10 toobtain one or more numbers indicative of a selected spectral componentof specimen 36. As the spectral component numbers from specimen 36 arepresented to frame buffers 48, computer 52 selects only those pixelshaving the selected spectral component or components. The image that isgenerated for display 54 contains only those pixels having the desiredspectral components, i.e., only the desired characteristics such as aparticular chromosome or cell or undergoing a particular chemicalreaction that produces the selected spectral component or components.

Thus, flow cytometer 10 may examine a serial flow of samples todetermine the spectral components of a particular sample of interest.These spectral components are communicated to computer 52 and thereafterused to form the image that is displayed from image cytometer 20 asspecimen 36 is scanned by laser beam 38. As a sample is scanned, thosepixels having the selected spectral components are enhanced inbrightness wherein all cells or subcellular structures with the desiredspectral properties appear bright, while all other details appear as adim background. This difference in intensity allows the identificationof those cells that belong to the subpopulation identified in flowcytometry and the analysis of their morphology.

This image enhancement is a "virtual sorting" of the sample componentshaving the selected spectral characteristics. It is equivalent in itsresults to physical sorting followed by microscopic examination, withthe major difference that, in the case of virtual sorting, one sortsnumerical data rather than actual cells. Apart from not requiring aphysical sorting capability on the flow cytometer, nor the collection ofsorted cells and the preparation of separate microscopy samples for eachsubpopulation of interest, virtual sorting allows large numbers ofsubpopulations to be analyzed simultaneously from a morphological pointof view. The number of simultaneously analyzed subpopulations is limitedonly by the number of channels in the data processing system of the FTimaging cytometer and by the spectral resolution of the FT spectrometers(typically eight or more), and can exceed by far the number ofsubpopulations that can be simultaneously physically sorted (typicallytwo to four).

In a "reversed" virtual sorting, the spectral properties ofmorphologically interesting cells are communicated from image cytometer20 to flow cytometer 10 to program the FT flow cytometer. In this case,the flow cytometer would rapidly provide population-level data on theabundance of the cells or cellular components of interest. The spectralproperties can also be used to program an optical trapping system, suchas taught by U.S. Pat. No. 4,887,721, issued Dec. 19, 1989, to separatecells identified as having the same spectral properties as thesubpopulation of interest. This technique, an "indirect sorting", isuseful when the staining properties of a subpopulation of rare cells areonly approximately known, but can be accurately and rapidly determinedin flow. Then, the imaging cytometer can accurately discriminate betweenthe rare cells of interest and the rest of the sample, and the automatedoptical trapping system can scan through the sample, identify the cells,and separate them.

The foregoing description of the preferred embodiments of the inventionhave been presented for purposes of illustration and description. It isnot intended to be exhaustive or to limit the invention to the preciseform disclosed, and obviously many modifications and variations arepossible in light of the above teaching. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical application to thereby enable others skilled in the art tobest utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

What is claimed is:
 1. An integrated fluorescence analysis system,comprising:flow cytometer means having birefringent optics for measuringa first plurality of simultaneous spectral wavelengths present in afirst fluorescence spectrum from a first sample; first processor meansfor resolving said first fluorescence spectrum into first numbersrepresenting the intensity of spectral components of said firstplurality of spectral wavelengths; an imaging cytometer havingbirefringent optics for measuring a second plurality of simultaneousspectral wavelengths present in a second fluorescence spectrum from asecond sample; second processor means for resolving said secondfluorescence spectrum into second numbers representing the intensity ofspectral components of said second plurality of spectral wavelengths;and means connecting said first processor means with said secondprocessor means for inputting said first numbers to said secondprocessor and said second numbers to said first processor forrespectively enhancing spectral components of said second and said firstsamples.
 2. An integrated fluorescence analysis system according toclaim 1, further including:scanning means for measuring said secondplurality of spectral wavelengths at discrete pixel locations over asurface of said second sample; and computer display means for generatingan image of said pixels having said second numbers enhanced by saidfirst numbers from said flow cytometer.
 3. An integrated fluorescenceanalysis system according to claim 1, wherein said imaging cytometerfurther includes:microscope means for scanning a laser beam over aselected sample image plane of said second sample to excite said secondfluorescence spectrum over said surface; birefringent optics forresolving said second fluorescence spectrum into a plurality of pixels,each pixel having a pixel fluorescence with one or more spectralwavelengths; wherein said second processor resolves each said pixelfluorescence into said second numbers representing the intensity ofspectral components is said pixel fluorescence; storage means forstoring said first number representing the intensity of spectralcomponents from said first spectrum; and; means for selecting pixelshaving numbers corresponding to said stored second numbers to develop animage over said image plane corresponding to only pixels with said firstspectrum.
 4. An integrated fluorescence analysis system according toclaim 3, wherein said microscope means is a confocal microscope.